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Production of 6'-o-sialyllactose and intermediates

a technology intermediates, which is applied in the field of oligosaccharides and derivatives, can solve the problems of unfavorable steric and electronic effects of stereoselective, inability to access large volumes of sialylated human milk oligosaccharides, and inability to scale up methodologies

Inactive Publication Date: 2013-02-07
GLYCOM AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention relates to a method for preparing 6′-O-sialyllactose or its salt, which involves several steps and the use of specific chemicals. The invention also provides novel compounds and salts of 6′-O-sialyllactose. The technical effects of this invention include improved methods for preparing a valuable nutritional and pharmaceutical additive, as well as novel compounds for use in nutritional and pharmaceutical compositions.

Problems solved by technology

To date, access to large volumes of sialylated human milk oligosaccharides has not been possible by using isolation, biotechnology and synthetic methodologies.
The chemical synthesis of sialylated human milk oligosaccharides is one of the most challenging fields of carbohydrate chemistry due to the nature of sialic acid donors themselves.
In general, stereoselective glycosylations are achieved via neighbouring group participations but the lack of a substituent at C-3 position of sialic acid prevents such an option.
Furthermore, the presence of the carboxylic moiety at the anomeric position of sialic acid also creates unfavoured steric and electronic effects for stereoselective α-sialylations.
However, these methodologies have not been attractive for scale-up due to lack of efficient purification methodologies, use of expensive and toxic reagents and the involvements of many synthetic steps.
The use of either very expensive or very toxic chemicals for the sialylation such as mercury cyanide, mercury bromide and silver carbonate is one of the reasons that make these methodologies less attractive for scale-up studies and production.
Non efficient stereocontrol and / or yields likewise make(s) the strategies less suitable for large scale technology developments.
Additionally, severe purification difficulties characterize all the listed synthetic strategies.
These complex enzymatic systems represent very expensive methodologies for scale up productions of 6′-O-sialyllactose.
Similarly, sialidases could not be used successfully in large scale production methodologies due to their lack of regio- and stereoselectivity.
Low yields and difficult purification protocols are likewise a hindrance for industrial scale technology developments.
The isolation of 6′-SL from human and other mammals' milk is rather difficult even in milligram quantities due to the presence of a large number of similar oligosaccharides.
Such methodologies have serious drawbacks in regulatory processes due to limiting commercialisation opportunities.

Method used

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  • Production of 6'-o-sialyllactose and intermediates
  • Production of 6'-o-sialyllactose and intermediates
  • Production of 6'-o-sialyllactose and intermediates

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0218]

[0219]A mixture of anhydrous sialic acid (100 g, 323 mmol) and dried Amberlite IR-120 (H+) ion exchange resin (100 g) in MeOH (1500 mL) was stirred for 15 hours at RT. The ion exchange resin was filtered off and washed with MeOH (2×100 mL). The washes were combined with the filtrate and concentrated to 300 mL. The concentrated residue crystallized upon seeding at RT. The crystals were collected by filtration giving 73.8 g (71%) sialic acid methyl ester. The mother liquor was concentrated (10.5 g) and recrystallized from MeOH (30 mL) to yield 7.4 g (7%) sialic acid methyl ester. Total yield 81.2 g (78%).

[0220]1H NMR (D2O) δ in ppm: 1.89 (dd, 1H, J=13.0 Hz, J=11.4 Hz); 2.03 (s, 3H); 2.28 (dd, 1H, J=13.0 Hz, J=4.7 Hz); 3.52 (dd, 1H, J=8.9 Hz, J=3.0 Hz); 3.59 (dd, 1H, J=11.6 Hz, J=6.1 Hz); 3.71 (ddd, J=8.9 Hz, J=2.5 Hz, J=11.6 Hz); 3.82 (dd, 1H, J=11.6 Hz, J=2.5 Hz); 3.82 (s, 3H); 3.90 (dd, 1H, J=10.1 Hz, J=10.0 Hz); 3.98-4.10 (m, 2H, H-6, H-4).

example 2

[0221]

[0222]To a suspension of anhydrous sialic acid (100 g, 323 mmol) in MeOH (1200 ml) [0.03% water content] 8% HCl in MeOH (50 ml) was added and the reaction mixture was stirred for 6 hours at RT. The reaction mixture was neutralized with triethylamine (15 ml) and the clear solution was concentrated to 270 mL. The concentrated residue crystallized upon seeding at RT for 2 hours. The solid was collected by filtration yielding 104.9 g (100%).

example 3

[0223]

[0224]A suspension of sialic acid methyl ester (50 g, 155 mmol) and acetic anhydride (73 ml, 775 mmol) in DCM (175 mL) was stirred at RT and 70% perchloric acid (1 mL) was then added dropwise within 30 minutes. During the addition the temperature of the mixture increased until reflux. The reaction mixture was stirred at reflux for 2.5 h, and after this time MeOH (7.5 ml, 185 mmol) was added dropwise and the reaction mixture was stirred for a further hour at RT. The reaction mixture was diluted with DCM (175 mL) and washed with water (3×50 mL). The combined water phases were extracted with DCM (2×100 mL). The combined organic phases were washed with saturated NaHCO3 (2×100 mL) and evaporated. The residue (59.6 g) was dissolved in iBuOAc at 50° C. and the mixture was cooled down to RT and let overnight complete the crystallization. The solid was collected by filtration yielding 39.2 g (52%) of tetraacetyl sialic acid methyl ester.

[0225]1H NMR(C6D6) δ in ppm: 1.60, 1.63, 1.70, 1....

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Abstract

The present invention relates to a method for preparation of the trisaccharide 6′-0-sialyllactose (formula (I)) or salts thereof as well as intermediates in the synthesis and for the use of 6′-0-sialyllactose salts in pharmaceutical or nutritional compositions.

Description

FIELD OF THE INVENTION[0001]The present invention provides novel oligosaccharides and derivatives thereof, and furthermore methods for the preparation of these oligosaccharides, especially in large scale.BACKGROUND OF THE INVENTION[0002]During the past decades the interest for preparation and commercialization of human milk oligosaccharides has been increasing steadily. The importance of human milk oligosaccharides is directly linked to their unique biological activities such as antibacterial, antiviral, immune system and cognitive development enhancing activities [1].[0003]Sialylated human milk oligosaccharides such as disialyllacto-N-tetraose, 3′-O-sialyl-3-β-fucosyllactose, 6′-O-sialyllactose, 3′-O-sialyllactose, 6′-O-sialylated-lacto-N-neotetraose, 3′-O-sialylated-lacto-N-tetraose etc. are major components of human milk.[0004]Among the above listed sialylated human milk oligosaccharides the sialic acid residue is always linked to the terminal 3′-O- and / or 6′-O— position(s) of D-...

Claims

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Application Information

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IPC IPC(8): C07H1/00C07H5/10C07H3/04C07H5/04
CPCA23L1/308C07H3/06A61K31/702A23L1/30C07H13/04C07H15/18C07H1/00A23L33/10A23L33/21A61P25/28A61P3/02A61P31/04A61P31/12A61P37/02
Inventor PEREZ FIGUEROA, IGNACIOHORVATH, FERENCDEKANY, GYULAGOSTON, KAROLYGOSTON, GNESBAJZA, ISTVANBOUTET, JULIENHEDEROS, MARKUSKOVACS-PENZES, PIROSKAKROGER, LARSROHRIG, CHRISTOPHSCHROVEN, ANDREASVRASIDAS, IOANNISRISINGER, CHRISTIAN
Owner GLYCOM AS
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